QC: Minimal intensity of infected cells

[tischi]

I measured some individual well where there was barely any IgA signal (and pipeline gave me a false positive) - if the signal mean of infected cells is below 300 (after background subtraction) than we can say "no IgA signal" (or something like that)... 300 is just an approximation from measuring few wells, it would be nice to be able to figure this better...also is "mean" a good value to go for here? I want to stress that the signal in "infected" cells is relevant and not the signal in "control" cells. Also, these wells should be labelled not with NaN but with "no IgA signal" because they are in principle negative. We should add the same threshold (300) for IgG channel (IgG is less prone for "no signal" situation, IgG was our focus more than IgA and that is why this QC didn't appear essential). Let me know what you think?

[tischi]

• [ ] Plot ratio score and z-score against mean intensity in infected cells for both IgG and IgA (4 plots)
• [ ] Extract measured cell intensities in infected cells in H01 wells.

[tischi]

IgG for one plate

The three dim ones include H01 and G01; they are all below 100; the crowd starts at around 800 on the x-axis.

[tischi]

IgA for one plate

The bulk starts around 200 on the x-axis

[tischi]

The highlighted row is H01. One thing would be to take N * mad of the intensities measured in well H01 as a threshold. Note: @constantinpape has a different method for measuring the intensities in well H01 that includes all pixels. That could also be used, however then we have be careful whether H01 contained virus or not. If there was virus, the mad could be higher, so we would exclude more images on such plates.

[tischi]

IgG mad (using all pixels) in H01 wells; mean = 100.41

   plate_name                          `median(background_IgG_mad, na.rm = T)`
   <chr>                                                                 <dbl>
 1 plate8rep1_20200425_162127_242                                         90.6
 2 plate9_2rep2_20200515_230124_149                                       88.0
 3 plate9_3rep1_20200513_160853_327                                      102. 
 4 plateK12rep1_20200430_155932_313                                       89.1
 5 plateK13rep1_20200430_175056_461                                       89.8
 6 plateK14rep1_20200430_194338_941                                      104. 
 7 plateK15rep1_20200502_134503_016                                      112. 
 8 plateK16rep1_20200502_154211_240                                       83.9
 9 plateK17rep1_20200505_115526_825                                      130. 
10 plateK18rep1_20200505_134507_072                                      152. 
11 PlateK19rep1_20200506_095722_264                                       94.3
12 PlateK20rep1_20200506_114059_490                                      155. 
13 PlateK21rep1_20200506_132517_049                                      138. 
14 plateK22rep1_20200509_094301_366                                      139. 
15 plateK23rep1_20200512_103139_970                                      117. 
16 plateK25rep1_20200512_123527_554                                      217. 
17 plateK26rep1_20200515_221809_658                                       90.9
18 plateT1rep1_20200509_114423_754                                        72.2
19 plateT2rep1_20200509_190719_179                                        81.3
20 plateT3rep1_20200509_152617_891                                       101. 
21 plateT4rep1_20200509_171215_610                                        85.0
22 plateT5rep1_20200512_143609_835                                        92.2
23 plateT6_20200513_105342_945                                            84.2
24 plateT7_20200513_131739_093                                            97.2
25 plateT8rep1_20200516_091304_432                                       105. 
26 plateU10rep1_20200521_140753_720                                       78.9
27 plateU11rep1_20200521_154049_516                                       64.1
28 plateU13_T9rep1_20200516_105403_122                                   114. 
29 plateU1rep1_20200519_102648_382                                        73.7
30 plateU2rep1_20200519_120202_377                                        75.1
31 plateU3rep1_20200519_134143_061                                        89.7
32 plateU4rep1_20200519_155958_647                                        70.4
33 plateU5rep1_20200519_173432_260                                        78.7
34 plateU6rep1_20200519_192153_222                                       128. 
35 plateU7rep1_20200519_210009_665                                        67.9
36 plateU8rep1_20200519_223701_819                                       124. 
37 plateU9rep1_20200520_215725_694                                        78.8
38 titration_plate_20200403_154849                                        61.6

IgA mad (using all pixels) in H01 wells; mean = 130.9281

   plate_name                          `median(background_IgA_mad, na.rm = T)`
   <chr>                                                                 <dbl>
 1 plate8rep1_20200425_162127_242                                         164.
 2 plate9_2rep2_20200515_230124_149                                       131.
 3 plate9_3rep1_20200513_160853_327                                       122.
 4 plateK12rep1_20200430_155932_313                                       118.
 5 plateK13rep1_20200430_175056_461                                       122.
 6 plateK14rep1_20200430_194338_941                                       124.
 7 plateK15rep1_20200502_134503_016                                       126.
 8 plateK16rep1_20200502_154211_240                                       118.
 9 plateK17rep1_20200505_115526_825                                       153.
10 plateK18rep1_20200505_134507_072                                       156.
11 PlateK19rep1_20200506_095722_264                                       143.
12 PlateK20rep1_20200506_114059_490                                       303.
13 PlateK21rep1_20200506_132517_049                                       128.
14 plateK22rep1_20200509_094301_366                                       145.
15 plateK23rep1_20200512_103139_970                                       122.
16 plateK25rep1_20200512_123527_554                                       125.
17 plateK26rep1_20200515_221809_658                                       147.
18 plateT1rep1_20200509_114423_754                                        116.
19 plateT2rep1_20200509_190719_179                                        121.
20 plateT3rep1_20200509_152617_891                                        132.
21 plateT4rep1_20200509_171215_610                                        119.
22 plateT5rep1_20200512_143609_835                                        111.
23 plateT6_20200513_105342_945                                            110.
24 plateT7_20200513_131739_093                                            131.
25 plateT8rep1_20200516_091304_432                                        119.
26 plateU10rep1_20200521_140753_720                                       113.
27 plateU11rep1_20200521_154049_516                                       119.
28 plateU13_T9rep1_20200516_105403_122                                    131.
29 plateU1rep1_20200519_102648_382                                        117.
30 plateU2rep1_20200519_120202_377                                        140.
31 plateU3rep1_20200519_134143_061                                        119.
32 plateU4rep1_20200519_155958_647                                        121.
33 plateU5rep1_20200519_173432_260                                        121.
34 plateU6rep1_20200519_192153_222                                        120.
35 plateU7rep1_20200519_210009_665                                        111.
36 plateU8rep1_20200519_223701_819                                        112.
37 plateU9rep1_20200520_215725_694                                        116.
38 titration_plate_20200403_154849                                         NA 

[tischi]

IgG score vs infected cell intensity

IgA score vs infected cell intensity

[tischi]

IgM background mad

5 plate1_IgM_20200527_125952_707                                         268.
7 plate2_IgM_20200527_155923_897                                         610.
9 plate5_IgM_20200528_094947_410                                         176.
11 plate6_IgM_20200528_111507_585                                         116.
14 plate8rep2_20200502_182438_996                                         268.

The plate plate2_IgM_20200527_155923_897 has a quite high value. Should I inspect in the PlateViewer? @imagirom @constantinpape